Isolation of individual cell populations from their parent organs such as liver, spleen, kidney, adrenals, and pancreas has been most intensely studied with respect to isolation of intact islets of Langerhans from the pancreas. A number of attempts have been made to obtain preparations of islets which can be used for transplantation into subjects, such as diabetics, whose own islets are not effectively functioning to secrete insulin. Isolation of, and injection of, the islets is, of course, highly preferable to whole organ transplantation. In addition to the obvious advantage of eliminating the 98-99% of the pancreas that is not required and can only cause complications, the individual islets can be more effectively preserved using low temperature and can be more effectively encapsulated, if desired, in order to minimize immune response.
Islets were first isolated from rodent pancreas by Lacy, P.E., et al, Diabetes (1967) 16:35-39. This method involved the use of collagenase, and the procedure was applied to the isolation of islets from human tissue by Gray, D.W.R., et al, Diabetes (1984) 33:1055-1061. In the Gray procedure, the tissue in the collagenase digestion mixture was further disrupted by aspiration through different sized needles. An earlier adaptation to human tissue was reported by Sharp, D.W., et al, Surgery (1975) 77:100-105, and this also involved physical disruption of the tissue. A method generic to mammalian pancreatic tissue which utilizes chopping or grinding of the partially digested pancreatic tissue before size separation by screening was described by Scharp, D.W., et al, Methods in Diabetes Research (1985), Vol. 1, Part C, pp. 225-243, John Wiley & Son.
More recently, Kneteman, N.M., et al, Transplantation Proceedings (1986) 18:182-185, described a method for perfusing human pancreas with a collagenase solution via the pancreatic duct, similar to the method developed by Horaguchi, A., et al, Diabetes (1981) 30:455-458. Subsequent treatment also involved disruption of the tissue. Kuhn, F., et al, Biomed Biochim Acta (1985) 44:149-153, while adopting the collagenase perfusion method employing the pancreatic duct, utilized Velcro strips in the digestion tube (duct) in order to retain the partially digested islet-containing tissue in a method adapted from that reported for the isolation of beef islets by Lacy, P.E., et al, Diabetes (1982) 31:109-111. Scharp, D.W., et al, in an abstract presented at American Diabetes meeting (Baltimore, June 1985) disclose a method for isolating human islets in which a collagenase solution injected into the pancreas was caused to distend the organ. The digested pancreas was passed then through a tissue macerator and size-segregated by screening.
All of the foregoing methods result in islet preparations which are useful, but which could be improved upon in terms of the quality of the islets obtained, as well as their purity. Possibly because these methods involve rather severe mechanical disruption of the tissue, the islets also become disrupted, and therefore less effective in secreting insulin. It has been calculated that one islet with a diameter of 350 microns contains more insulin-producing cells than 100 islets with an average diameter of 75 microns. Thus, more than 100 islets of the smaller size are needed for every individual islet of the larger diameter.
It would therefore be useful to provide a means to obtain islets of large diameter and high purity. These islets could then be more effectively stored, encapsulated, and transplanted to alleviate the symptomology associated with low insulin secretion. Similar considerations apply to preparation of liver cells, spleen cells, and so forth, for transplantation in connection with the relevant disorder.